﻿Lysimachiadanxiashanensis, a new species of Primulaceae from Guangdong, China

﻿Abstract Lysimachiadanxiashanensis, a new Primulaceae species, endemic to the Danxia landscape in Guangdong Province, China, is described and illustrated. This new species is morphologically similar to L.pseudohenryi, L.phyllocephala, L.congestiflora and L.kwangtungensis, but it differs from the similar species by its purplish-red plants, petiole without wings, calyx with orange glandular and the corolla margin serrated on upper half with orange-red glandular punctates. This new species belongs to Lysimachiasubgen.Lysimachiasect.Nummularia. Phylogenetic analysis confirmed that L.danxiashanensis is a distinct clade, based on the combined data of ITS and rbcL sequences. The conservation status of the new species was evaluated as Endangered (EN) according to IUCN Red List Categories and Criteria.


Introduction
Lysimachia L. is a member of the tribe Lysmachieae (Primulaceae) and is composed of over 200 species (Chen and Hu 1979;Hu and Kelso 1996;Wang et al 2018).This genus is the most widely distributed genera of Primulaceae and is mainly distributed in the temperate and subtropical regions of the Northern Hemisphere, but there are also a few species in Africa, Australia and South America (Hu 1994;Hao and Hu 2001;Hao et al. 2004;Kodela 2006).China is considered to be the origin and diversity centre of Lysimachia, with 138 native species having been recorded in Flora of China and nearly 80% of them are endemic species (Chen and Hu 1979;Hu and Kelso 1996;Hao and Hu 2001).In recent years, many new species of this genus have been described and this highlights more opportunities for discoveries in China (Wang et al 2018;Huang et al 2020;Yan et al 2022).
An unknown species of Lysimachia was discovered during a field floristic investigation from May 2022 to August 2023 in Danxiashan National Park, Renhua County, Guangdong Province.It is most similar to L. congestiflora Hemsl., but its purplish-red plants, petiole without wings, corolla lobes serrated on PhytoKeys 237: 257-268 (2024), DOI: 10.3897/phytokeys.237.114484 Xing-yue Zhang et al.: Lysimachia danxiaensis, a new species from China upper half and calyx with orange glandular punctates clearly distinguish from the latter.After careful morphological comparison by specimens and consultation with relevant literature and molecular phylogenetic analysis, we confirmed that it represented a new species, described and illustrated here.The threat status of the new species is assessed according to the IUCN Red List Categories and Criteria (IUCN Standards and Petitions Committee 2022).

Morphological study
The morphological characters of the new species were observed and measured, based on fresh and dry specimens using a micrometer and a stereomicroscope and were compared with its related species, based on herbarium specimens deposited at the Herbarium of SYS and IBSC (the herbarium acronyms follow Thiers ( 2023)), as well as the digital images on the Chinese Virtual Herbarium (https://www.cvh.ac.cn/) and the China Field Herbarium (https://www.cfh.ac.cn/).Morphological observation and examination were conducted in the SYS.

Taxon sampling and molecular analysis
Leaf tissue of the putative new species and related species was collected from one population and silica dried in zip-lock plastic bags until use for comparisons and taxonomic treatment.Total DNA was extracted with a modified CTAB method (Doyle and Doyle 1987).Regions of the partial internal transcribed spacer 1, the 5.8S ribosomal RNA gene and partial internal transcribed spacer 2 were amplified using the previously-reported primers ITS1 and ITS4 (White et al. 1990) and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene was amplified using the primers rbcLa-f (Kress and Erickson 2007) and 724R (Fay et al. 1997).PCR amplifications were performed following Fan et al. (2015).Following the studies of Lysimachia (Zhang et al. 2011;Yan et al. 2018), we retrieved 67 ITS and rbcL accession of 30 species from GenBank, which belong to subgenus Lysimachia L., subgenus Palladia (Moench) Hand.-Mazz., subgenus Heterostylandra (Hand.-Mazz.)Chen et C.M.Hu.and subgenus Idiophyton Hand.-Mazz.Two accessions of the putative new species (GenBank Acc.ITS No.: OR665389, OR665390; rbcL No: PP025352, PP035354) and one accession of Lysimachia kwangtungensis (GenBank Acc.ITS No.: OR941025; rbcL No: PP025355) were sequenced for this study.Ardisia verbascifolia was selected as outgroup.The sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) and subsequently manually adjusted.Phylogenetic constructions were based on Maximum Likelihood (ML) and Bayesian Inference (BI) and were respectively run by IQ-TREE v. 2.0.3 (Minh et al. 2020) and MrBayes version 3.1.2(Huelsenbeck and Ronquist 2001), selecting best-fit model as SYM+I+G4 with 2000 bootstraps (BS) for ML analysis.ModelFinder v.2.2.0 (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (Edge-linked) using the BIC criterion.The best-fit models according to BIC were SYM+G4 for ITS and K2P+I+G4 for rbcL.BI analysis employed random starting trees and four Markov Chain Monte Carlo (MCMC) simulations were run simultaneously and sampled every 1000 generations for 10 million generations.The average standard devi- PhytoKeys 237: 257-268 (2024) ation of split frequencies (< 0.01) was used to assess the convergence of the two runs.Bayesian posterior probabilities (PP) were calculated as the majority consensus of all sampled trees with the first 25% discarded as burn-in.

Morphological comparison
According to the classification of Chen and Hu (1979), L. danxiashanensis is a member of subgenus Lysimachia sect.Nummularia, which is characterised by stems prostrate to erect on the upper part, leaves opposite, racemes shortened to subcapitate, filaments longer than anthers, lower part connate into a tube, corolla and calyx with coloured glandular punctates (Fig. 2).In China, there are over 50 species of sect.Nummularia and it widely distributed from southwest to the east and south China.Morphologically, Lysimachia danxiashanensis is similar to L. phyllocephala Hand.-Mazz., L. pseudohenryi Pamp., L. congestiflora Hemsl.and L. kwangtungensis (Hand.-Mazz.)C.M.Hu by sharing the following morphological features: stems with multicellular hairs, leaves opposite and racemes terminal.However, the new species can be easily distinguished from similar species by combination characters including its purplish-red plants (vs.green), petiole without wings (vs.narrowly winged in L. pseudohenryi and L. congestiflora, narrowly margined and auriculate at base in L. kwangtungensis and absent in L.phyllocephala) and the corolla lobes serrated on upper half (vs.entire margin in all four species).A more detailed morphological comparison of these species is summarised in Table 1.

Molecular analysis
The combined aligned matrix had a length of 1268 bp (ITS: 650, rbcL: 615), including 373 variable sites, of which 291 were parsimony-informative.The two accessions of the new species were from the same population and formed a separate monophyletic lineage (Fig. 1: BS = 93%, PP = 0.72), the sister group of L. rubiginosa.Although L. danxiashanensis and L. rubiginosa both belong to subgen.Lysimachia sect.Nummularia, the new species can be easily distinguished from the latter by its shorter plants (10-28 cm vs. 30-60 (100) cm), orange glandular punctates on corolla lobes and calyx (vs.black or brown glandular striate on leaves, corolla lobes and calyx), 5-9 flowered on branches and stems axis (vs.3-5 flowered on branches, seldom on main axis).Geographically, L. rubiginosa is distributed in Guangxi, Guizhou, Hubei, Hunan, Sichuan, Yunnan and Zhejiang and it mainly grows in limestone.In contrast, the new species is distributed in Danxia landscape, Guangdong.The geographical distribution of these two taxa does not overlap.
Although the infrageneric phylogenetic relationships within Chinese Lysimachia remain controversial (Zhen and Chen 2012;Liu et al. 2023), the phylogenetic tree placed L. danxiashanensis distant from other species in this genus (Fig. 1).Based on the morphological and molecular evidence, we confirmed that L. danxiashanensis is a distinct species.Therefore, we describe and provide illustrations for the new species below.Diagnosis.Lysimachia danxiashanensis can be distinguished from L. congestiflora by its purple-red plants (vs.green), petiole without wings (vs.narrowly winged), corolla lobes yellow with serrations on upper half (vs.dull red at base with entire margin) and calyx with orange glandular (vs.without glandular) (Fig. 4).
Phenology.The flowering of Lysimachia danxiashanensis is from May to June; and the fruiting in June.
Distribution.Lysimachia danxiashanensis is currently known only from the type locality, Danxiashan National Nature Reserve, Guangdong, China.
Habit.Lysimachia danxiashanensis was observed to grow on wet rocks of Danxia landform at elevations 270 to 320 m.
Etymology.The specific epithet refers to the type locality, Danxiashan National Nature Reserve in Guangdong Province, China.
Local name.The Chinese name of the new species is here given as 丹霞山过 路黄 (Dān xiá Shān Guò Lù Huáng).
Provisional conservation status.Endangered (EN).In the past two years, we have conducted several field investigations on the Danxia landscapes in Guangdong Province, with only four populations of Lysimachia danxiashanensis being found in Danxiashan National Nature Reserve and the number of mature individuals is stable (total < 200 individuals); However, its habitat is on the side of the road, which is vulnerable to human disturbance.L. danxiashanensis

Figure 1 .
Figure 1.Phylogenetic tree of Lysimachia danxiashanensis and related species generated by Bayesian Inference (BI) of the combined dataset (ITS, rbcL).Bootstrap values of the ML and BI posterior probabilities are shown along the branches.The new species in yellow shaded area, green indicates subgen.Lysimachia, orange is subgen.Palladia, purple is subgen.Heterostylandra and grey is subgen.Idiophyton.Blue indicates outgroup, Ardisia verbascifolia.

Figure 2 .
Figure 2. Lysimachia danxiashanensis W.B.Liao, Q.Fan & G.D.Tang, sp.nov.A habit B flowering branch C inflorescence D abaxial and adaxial views of leave E lateral view of flower F dorsal view of flower G adaxial side of corolla lobes H stamens I immature capsule J abaxial (1 st , 2 nd , 5 th ) and adaxial (3 rd , 4 th ) views of calyx lobes K pistil and densely pilose pedicel L stigma M cross-section of ovary (Photographers: A, C, D, I by Xing-Yue Zhang; B, E, F by Qiang Fan; G, H, J-M by Jing-Min Dai).

Figure 3 .
Figure 3. Lysimachia danxiashanensis W.B.Liao, Q.Fan & G.D.Tang, sp.nov.A habit B abaxial (left) and adaxial (right) views of leaf C adaxial side of corolla lobes D lateral view of flower E dorsal view of flower F stamens G pistil and stigma H fruit I adaxial (left) and abaxial (right) views of calyx (Drawn by Rong-En Wu).